The present invention relates to the field of detection and enrichment of a desired nucleic acid from a population of nucleic acids in a sample, especially the enrichment of rare nucleic acids containing mutations.
Single nucleotide polymorphisms (SNPs) are the most common type of variation in the human genome. Point mutations are also usually SNPs but the term mutation is normally reserved for those SNPs with a frequency rarer than 1% and/or where there is a known correlative or functional association between the mutation and a disease (Gibson N J, 2006 Clin Chim Acta. 363(1-2):32-47). There are many applications for genotyping polymorphisms and detecting rare mutations. Rare variant detection is important for the early detection of pathological mutations, particularly in cancer. For instance, detection of cancer-associated point mutations in clinical samples can improve the identification minimal residual disease during chemotherapy and detect the appearance of tumour cells in relapsing patients. The detection of rare point mutations is also important for the assessment of exposure to environmental mutagens, to monitor endogenous DNA repair, and to study the accumulation of somatic mutations in aging individuals. Additionally, more sensitive methods to detect rare variants can revolutionise prenatal diagnosis, enabling the characterisation of foetal cells present in maternal blood. A vast number of methods have been introduced, but no single method has been widely accepted. Many methods for detecting low-frequency variants in genomic DNA use the polymerase chain reaction (PCR) to amplify mutant and wild-type targets. The PCR products are analysed in a variety of ways, including sequencing, oligonucleotide ligation, restriction digestion, mass spectrometry or allele-specific hybridization to identify the variant product against a background of wild-type DNA. Other methods use allele-specific PCR to selectively amplify from the low-frequency variant, with or without additional selection. For example, by digesting PCR products with a restriction enzyme that specifically cleaves the wild-type product. Current approaches have inherent limitations due to the lack of total specificity of allele-specific primers during PCR, which creates false positives. As a result, all current approaches have limited sensitivity and accuracy (review in Jeffreys A J and May C A, 2003 Genome Res. 13(10):2316-24).
Most mutation detection systems yield an assay signal that is difficult to validate in terms of the number of mutant molecules detected. This can be overcome in part by analyzing multiple samples, each containing limited DNA (typically 50 genome equivalents), to determine the number of mutant molecules in the sample. (digital PCR; Vogelstein and Kinzler 1999 Proc Natl Acad Sci USA. 96(16):9236-41). However, the large number of PCR reactions required, combined with background noise arising from misincorporation of nucleotides during PCR is likely to limit this approach to detection levels of about 1 variant in a population of 1000 nucleic acids. Another limitation of many mutation detection procedures is that they replace the mutant site with a PCR primer sequence and yield short amplicons containing little, if any, information other than the presence of a putative mutant allele (review in Jeffreys A J and May C A, 2003).
The unifying problem behind all of these PCR approaches for detecting rare variants is replication infidelity during amplification. Jeffreys and May have provided a solution by enriching mutant DNA molecules from genomic DNA prior to analyzing them by PCR; a process called DNA enrichment by allele-specific hybridization (DEASH) (Genome research 13:2316-2324, 2003). This method is a modification of traditional nucleic acid-enriching techniques that utilise hybridization with biotinylated DNA probes. It uses allele-specific oligonucleotides to fractionate DNA molecules differing by a single base substitution. However, this method of DNA enrichment involves multiple steps, requires large amounts of starting material and suffers from low sensitivity and efficiency.
Another enriching method is based on Restriction Fragment Length Polymorphism (RFLP), where PCR-amplified products are digested with restriction enzymes that can selectively digest either a normal or a mutated allele. Enriched PCR is a modification introduced into the RFLP analysis. The principle of this approach is to create a restriction enzyme site only within normal sequences, thus enabling selective digestion of the normal alleles amplified in a first amplification step. This prevents the non-mutant DNA from further amplification in a second amplification step while, upon subsequent amplification, the mutated alleles are enriched (U.S. Pat. No. 5,741,678; Kahn et al, 1991). This approach is limited, however, to the analysis of mutations at precise locations where restriction enzyme sites naturally occur. To overcome this limitation, one can artificially introduce restriction enzyme sites near the site of the point mutation to distinguish between normal and mutant alleles. In this approach, base-pair substitutions are introduced into the primers used for the PCR, yielding a restriction enzyme site only when the primer flanks a specific point mutation. This approach enables the selective identification of a point mutation at a known site, presumably in any gene.
Mismatched 3′ end amplification is a PCR technique which utilizes primers that have been modified at the 3′ end to match only one specific point mutation. This method relies on conditions which permit extension from primers with 3′ ends complementary to specific mismatches, whereas wild-type sequences are not extended. This procedure requires specific primers for each mutation and the PCR conditions are quite rigorous.
Recently, enrichment methods called PNA (or LNA) clamp PCR have been developed. High affinity nucleic acid analogues such as peptide-nucleic acids (PNAs) are used to inhibit nucleic acid amplification (U.S. Pat. No. 5,891,625). These methods can be problematic, however. It is difficult to find the optimal conditions for the PNA/LNA clamp; lengthy testing and redesigning are often required, and the purchase of specialised instruments may be needed. Furthermore, PNAs are expensive and difficult to synthesise and the efficiency of inhibition is often low.
EP1061135 relates to methods for detecting and identifying sequence variations in a nucleic acid sequence of interest using a detector primer. The publication concerns utilises diagnostics mismatches between the detector primer and the target where it occurs. The detector primer hybridizes to the sequence of interest and is extended with polymerase. The efficiency of detector primer extension is generally directly detected as an indication of the presence and/or identity of the sequence variation in the target
Nevertheless, it will therefore be appreciated that the provision of novel methods and probes adapted for sensitive enrichment and detection of rare point mutations would be a contribution to the art.